Carcinoembryonic antigen (hereinafter abbreviated as "CEA") is a glycoprotein discovered in 1965 by Gold and Freedman as an antigen which is found in both human colon cancer and the digestive organs of a 2-6 months old human embryo and having a molecular weight of 180,000 to 200,000 [Gold, P. & Freedman, S. O., J. Exp. Med., 121, 439 (1965)]. Its usefulness in cancer clinic has been widely recognized. It is a tumor marker which is used most often these days.
Further, it became evident by subsequent research that substances similar to CEA, that is, CEA-related antigen also exists in normal human tissues.
The term "CEA-related antigen" is a generic term for a group of antigens which are extremely similar to CEA in both proteinchemistry and immunology. Known typical CEA-related antigens include a nonspecific cross-reactive antigen which is a glycoprotein found in the lungs and spleen of a normal human being and having a molecular weight of about 90,000 [hereinafter abbreviated as "NC"; von Kleist, S. et al., Proc. Natl. Acad. Sci. U.S.A., 69, 2492 (1972)], NCA-2 found in embryonic feces [Burtin, P. et al., J. Immunol., 111, 1926 (1973)], and NFA (normal fecal antigen) found in normal adult feces [Kuroki, M. et al., Cancer Res. 41, 713 (1981)].
They are however still unknown in many aspects, including their detailed molecular structures and their differences in molecular structure.
Keeping step with changes in the diets of Japanese in recent years, colon cancer patients are increasing. About 80% of colon cancer is said to metastasize to the liver, and colon cancer is also said to metastasize to other organs such as the lungs. Recently, there has also been reported the possibility that the cell adhesion activity of CEA may take part in the metastasis of colon cancer to the liver [Jessup, J. M. et al., Cancer and Metastasis Reviews, 8, 263 (1989)].
Using a molecular biological technique, the primary structure of peptide has recently been ascertained with respect to CEA [Oikawa, S. et al., B.B.R.C., 142, 511 (1987) and Japanese Patent Laid-Open No. 177794/1988]. NCA [Tawaragi, Y. et al., B.B.R.C., 150, 89 (1988)], BGPI [Hinoda, Y. et al., Proc. Natl. Acad. Sci. U.S.A., 85, 6959 (1988)] and W272 (CGM6) [Arakawa, F. et al., B.B.R.C., 166, 1063 (1990)].
According to the results of a cDNA analysis, CEA peptide is composed of 668 amino acids as shown, for example, in FIG. 3 of Japanese Patent Laid-Open No. 177794/1988.
As is evident from the figure, CEA peptide can be divided into five domains, that is, domain N (1-108) extending from the N terminal to the 108th amino acid, domain I (109-286), domain II (287-464) and domain III (465-642) having mutually very homogeneous repetitive structures and composed individually of 178 amino acids, and domain M (643-668) composed of twenty-six, primarily hydrophobic amino acids on the side of the C terminal. Domains I, II and III may each be divided further into sub-domains (which may also be called "domains") which consist of 92 amino acid residues and 86 amino acid residues, respectively. These individual sub-domains may also be referred to as "1A", "1B", "2A", "2B", "3A" and "3B", respectively.
These individual domains and CEA peptide constructed of them are schematically illustrated at the top in FIG. 1 of Japanese Patent Application No. 222379/1991.
Domain M was first considered to be anchored on a cell membrane. It is however known that, after translation, domain M is processed and PI-G (phosphatidylinositol glycan) is added instead and is anchored on a cell membrane [Hefta, S. A. et al., Proc. Natl. Acad. Sci. U.S.A., 85, 4648 (1988); Takami N. et al., J. Biol. Chem., 263, 12716 (1988)].
All 12 cysteine residues are found in domains I, II and III, each domain having 4 residues at the same positions as others. Two loops are formed, one being located between the first cysteine and the second cysteine and consisting of 47 amino acids and the other between the third cysteine and the fourth cysteine and 39 amino acids, whereby CEA is considered, as a whole, to have a shape in which six loops are held.
NCA, on the other hand, contains domain N consisting of 108 amino acids and domain I consisting of 178 amino acids. Portions corresponding to domains II and III of CEA are however not found and, in continuation with domain I, there is domain M composed of 24 amino acids which are primarily hydrophobic. The structure of domain I of NCA shows homology slightly lower than 90% to that of CEA in terms of amino acids and the positions of the four cysteine residues contained therein are exactly the same. BGPI is a CEA-related antigen which is found in bile [Svenberg, T. et al., Int. J. Cancer, 17, 588 (1976)]. By a cDNA analysis, BGPI has been found to contain domain A', transmembrane domain and a cytoplasmic domain, which are characteristic to BGPI, after domain N and domain I.
W272 is a CEA-related antigen isolated from human leukocytes (granulocytes) and has a domain structure similar to NCA.
Biological activities of the CEA family have long remained unknown but, recently, CEA and NCA have been found to have cell adhesion activity [Oikawa, S. et al., B.B.R.C., 164, 39 (1989); Benchimol, S. et al., Cell, 57, 327 (1989)]. Namely, it has been found that cells with both CEA and NCA expressed on their surfaces firmly adhere to each other and also that cells with CEA expressed thereon and those with NCA expressed thereon strongly adhere to each other.
However, it has not been ascertained in which regions of CEA and NCA cell adhesion activity is present. This has remained as a bottleneck for the efficient production of a monoclonal anti-CEA antibody which inhibits the cell adhesion activity of CEA.
Any monoclonal antibody against the whole CEA molecule unavoidably contains many antibodies which in turn recognize peptides as epitopes other than the region (domain) having the cell adhesion activity of CEA. To permit efficient production of a monoclonal antibody capable of inhibiting the cell adhesion activity of CEA, it has been necessary to identify the region having the cell adhesion activity of CEA and also to determine its minimum peptide unit.